The development of vaccines to combat vaccine resistant cancers and infectious diseases has relied significantly on constructs employing subunit antigens. While the use of defined molecular antigens offers advantages in terms of safety and precision in immune response targeting, they are typically less immunogenic. Often, these vaccine formulations require an adjuvant, a substance that potentiates immune response. The critical roles of vaccine adjuvants lie in their ability to (1) enable the use of otherwise impotent antigens, (2) extend the benefits of vaccines to poor responders (e.g., older or immune-compromised patients), and (3) effect dose-sparing of rare and expensive antigens in short supply (e.g., in an epidemic). The natural product saponin QS-21 is the adjuvant of choice in numerous promising antitumor and antiviral vaccine clinical trials, despite liabilities associated with toxicity, chemical instability, and lack of availability in pure form. This grant application comprises a unique multi-disciplinary approach toward the preparation, discovery, evaluation, and understanding of potent adjuvant molecules essential in the development of vaccines for cancer and infectious diseases. The Specific Aims of this research program will involve: (1) the development of novel and efficient strategies for the chemical synthesis of designed non-natural structural variants of the saponin QS-21 possessing improved activity and stability with attenuated toxicity; (2) the evaluation of these novel compounds in mice to assess their ability to potentiate immune response, as well as their specific toxicity profiles; and (3) to employ small animal radio-imaging technologies, immunohistology, cytokine profiling, and affinity chromatography to gain insight into the adjuvant's as yet unknown mechanism of action. These efforts will address fundamental chemical and immunological questions associated with this extremely complex class of adjuvants, and holds the promise of yielding novel therapeutics in the vaccine arena.